Lys-70 carboxylation in the active site of class D β lactamases is essential for their activity. Structural, kinetic and affinity studies show that this post-translational modification can be affected by ... [more ▼]

Lys-70 carboxylation in the active site of class D β lactamases is essential for their activity. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val-117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys 70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate limiting step for the wild type OXA 10 β lactamase. [less ▲]

Lysozyme from lambda bacteriophage (lambda lysozyme) is an 18 kDa globular protein displaying some of the structural features common to all lysozymes; in particular, lambda lysozyme consists of two ... [more ▼]

Lysozyme from lambda bacteriophage (lambda lysozyme) is an 18 kDa globular protein displaying some of the structural features common to all lysozymes; in particular, lambda lysozyme consists of two structural domains connected by a helix, and has its catalytic residues located at the interface between these two domains. An interesting feature of lambda lysozyme, when compared to the well-characterised hen egg-white lysozyme, is its lack of disulfide bridges; this makes lambda lysozyme an interesting system for studies of protein folding. A comparison of the folding properties of lambda lysozyme and hen lysozyme will provide important insights into the role that disulfide bonds play in the refolding pathway of the latter protein. Here we report the (1)H, (13)C and (15)N backbone resonance assignments for lambda lysozyme by heteronuclear multidimensional NMR spectroscopy. These assignments provide the starting point for detailed investigation of the refolding pathway using pulse-labelling hydrogen/deuterium exchange experiments monitored by NMR. [less ▲]

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in combination with quenched-flow hydrogen exchange labeling, monitored by two-dimensional NMR and electrospray ionization mass spectrometry, to investigate the folding kinetics of lysozyme from bacteriophage lambda (lambda lysozyme) at pH 5.6, 20 degrees C. The first step in the folding of lambda lysozyme occurs very rapidly (tau < 1 ms) after refolding is initiated and involves both hydrophobic collapse and formation of a high content of secondary structure but only weak protection from (1)H/(2)H exchange and no fixed tertiary structure organization. This early folding step is reflected in the dead-time events observed in the far-UV CD and ANS fluorescence experiments. Following accumulation of this kinetic molten globule species, the secondary structural elements are stabilized and the majority (ca. 88%) of refolding molecules acquire native-like properties in a highly cooperative two-state process, with tau = 0.15 +/- 0.03 s. This is accompanied by the acquisition of substantial native-like protection from hydrogen exchange. A double-mixing experiment and the absence of a denaturant effect reveal that slow (tau = 5 +/- 1 s) folding of the remaining (ca. 12%) molecules is rate limited by the cis/trans isomerization of prolines that are trans in the folded enzyme. In addition, native state hydrogen exchange and classical denaturant unfolding experiments have been used to characterize the thermodynamic properties of the enzyme. In good agreement with previous crystallographic evidence, our results show that lambda lysozyme is a highly dynamic protein, with relatively low conformational stability (DeltaG degrees (N-U) = 25 +/- 2 kJ.mol(-1)). [less ▲]

Class A beta-lactamases (M(r) approximately 29000) provide good models for studying the folding mechanism of large monomeric proteins. In particular, the highly conserved cis peptide bond between residues ... [more ▼]

Class A beta-lactamases (M(r) approximately 29000) provide good models for studying the folding mechanism of large monomeric proteins. In particular, the highly conserved cis peptide bond between residues 166 and 167 at the active site of these enzymes controls important steps in their refolding reaction. In this work, we analyzed how conformational folding, reactivation, and cis/trans peptide bond isomerizations are interrelated in the folding kinetics of beta-lactamases that differ in the nature of the cis peptide bond, which involves a Pro167 in the BS3 and TEM-1 enzyme, a Leu167 in the NMCA enzyme, and which is missing in the PER-1 enzyme. The analysis of folding by spectroscopic probes and by the regain of enzymatic activity in combination with double-mixing procedures indicates that conformational folding can proceed when the 166-167 bond is still in the incorrect trans form. The very slow trans --> cis isomerization of the Glu166-Xaa167 peptide bond, however, controls the final step of folding and is required for the regain of the enzymatic activity. This very slow phase is absent in the refolding of PER-1, in which the Glu166-Ala167 peptide bond is trans. The double-mixing experiments revealed that a second slow kinetic phase is caused by the cis/trans isomerization of prolines that are trans in the folded proteins. The folding of beta-lactamases is best described by a model that involves parallel pathways. It highlights the role of peptide bond cis/trans isomerization as a kinetic determinant of folding. [less ▲]